Endothelial Glycocalyx-Mediated Nitric Oxide Production in Response to Selective AFM Pulling
Endothelial Glycocalyx-Mediated Nitric Oxide Production in Response to Selective AFM Pulling
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DOI：
10.1016/j.bpj.2017.05.033
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发表时间：
2017-07-11
影响因子：
3.4
通讯作者：
Tarbell, John M.
中科院分区：
文献类型：
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作者：
Bartosch, Anne Marie W.;Mathews, Rick;Tarbell, John M.
一氧化氮（NO）是血管系统中的一种调节分子，由于内皮损伤而对其产生的抑制会导致心血管疾病。糖萼是哺乳动物上皮细胞表面上的糖脂、糖蛋白和蛋白聚糖的薄层。胞外力通过糖萼传递，启动胞内信号传导途径。在内皮细胞（EC），以前的研究表明，糖萼是一个显着的调解人的NO生产;内皮糖萼层（EGL）的降解显着减少EC生产的NO响应流体剪切应力。然而，这一过程中涉及的具体EGL成分尚未得到很好的确定。最近的工作，在体外使用短发夹RNA的方法表明，蛋白聚糖磷脂酰肌醇蛋白聚糖-1，而不是syndecan-1，是主要的核心蛋白介导剪切诱导NO的生产。我们利用原子力显微镜（AFM）选择性地施加力的融合大鼠脂肪垫EC（RFPEC）的EGL的组件，包括蛋白多糖和糖胺聚糖，观察每个组件如何单独有助于力诱导的NO的生产。4，5-二氨基荧光素二乙酸酯，一种细胞可渗透的荧光分子，被用来检测细胞内NO生产的变化。抗体包被的AFM探针表现出较强的表面结合RFPEC单层，与100-300 pN的平均粘附力。AFM拉动磷脂酰肌醇蛋白聚糖-1和硫酸乙酰肝素10分钟导致NO的产生显着增加，而拉动syndecan-1，CD 44，透明质酸，并与对照探针没有。我们的结论是，原子力显微镜拉可用于激活EGL介导的NO生产和硫酸乙酰肝素蛋白聚糖磷脂酰肌醇蛋白聚糖-1是一个主要的机械传感器剪切诱导NO生产。
Nitric oxide (NO) is a regulatory molecule in the vascular system and its inhibition due to endothelial injury contributes to cardiovascular disease. The glycocalyx is a thin layer of glycolipids, glycoproteins, and proteoglycans on the surface of mammalian epithelial cells. Extracellular forces are transmitted through the glycocalyx to initiate intracellular signaling pathways. In endothelial cells (ECs), previous studies have shown the glycocalyx to be a significant mediator of NO production; degradation of the endothelial glycocalyx layer (EGL) drastically reduces EC production of NO in response to fluid shear stress. However, the specific EGL components involved in this process are not well established. Recent work using short-hairpin RNA approaches in vitro suggest that the proteoglycan glypican-1, not syndecan-1, is the dominant core protein mediating shear induced NO production. We utilized atomic force microscopy (AFM) to apply force selectively to components of the EGL of confluent rat fat pad ECs (RFPECs), including proteoglycans and glycosaminoglycans, to observe how each component individually contributes to force-induced production of NO. 4,5-diaminofluorescein diacetate, a cell-permeable fluorescent molecule, was used to detect changes in intracellular NO production. Antibody-coated AFM probes exhibited strong surface binding to RFPEC monolayers, with 100-300 pN mean adhesion forces. AFM pulling on glypican-1 and heparan sulfate for 10 min caused significantly increased NO production, whereas pulling on syndecan-1, CD44, hyaluronic acid, and with control probes did not. We conclude that AFM pulling can be used to activate EGL-mediated NO production and that the heparan sulfate proteoglycan glypican-1 is a primary mechanosensor for shear-induced NO production.